INSULIN SUPPRESSES BILE-ACID SYNTHESIS IN CULTURED RAT HEPATOCYTES BYDOWN-REGULATION OF CHOLESTEROL 7-ALPHA-HYDROXYLASE AND STEROL 27-HYDROXYLASE GENE-TRANSCRIPTION

Evidence from in vivo studies indicates that the bile acid pool and bi le acid excretion are increased in humans with diabetes mellitus acid in experimental diabetic animals, and that both parameters return to n ormal levels after administration of insulin. To investigate the bioch emical background of these changes, the effects of insulin on bile aci d synthesis and cholesterol 7 alpha-hydroxylase and sterol 27-hydroxyl ase, two key enzymes in routing of cholesterol toward bile acids, were studied in cultured rat hepatocytes. Mass production of bile acids wa s dose dependently diminished, showing significant reduction (-33% to -53%) at physiological concentrations of the hormone (1.4 to 14 nmol/L ) and a maximal decrease at 140 nmol/L (-65%). The decrease of bile ac id synthesis correlated well with the suppression of cholesterol 7 alp ha-hydroxylase and sterol 27-hydroxylase activity. The enzyme activity for cholesterol 7 alpha-hydroxylase, examined in more detail, was dos e dependently diminished on incubation of hepatocytes with various con centrations of insulin, reaching maximal reduction at 14 nmol/L of ins ulin. Maximal decrease of the enzyme activity was seen after 8 hours o f incubation (-70%). Insulin strongly reduced the rise in cholesterol 7 alpha-hydroxylase activity induced by incubation with dexamethasone, Sterol 27-hydroxylase activity was inhibited up to -58% after 24 hour s of incubation with 140 nmol/L, insulin, To study the mechanism of su ppression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase activity, the effects of insulin on their respective levels of messen ger RNA (mRNA) and gene transcription were assessed. The decrease in e nzyme activities could be explained by a concomitant reduction in the cholesterol 7 alpha-hydroxylase (-76%) and sterol 27-hydroxylase (-62% ) mRNA level. Transcriptional activity, as assessed by nuclear runoff assays, was decreased to the same extent, i.e., -60% for cholesterol 7 alpha-hydroxylase and -75% for sterol 27-hydroxylase. Transient expre ssion experiments using a construct containing the proximal 348 basepa irs of the cholesterol 7 alpha-hydroxylase promoter fused to the chlor amphenicol acetyltransferase (CAT) gene (-348Rcat) showed a significan t reduction of transcriptional activity (-64%) with insulin, indicatin g that a sequence important for an insulin-induced transcriptional res ponse is located within the first 348 basepairs, preceding the transcr iption start of the cholesterol 7 alpha-hydroxylase promoter. We concl ude that physiological concentrations of insulin suppress bile acid sy nthesis by downregulation of cholesterol 7 alpha-hydroxylase and stero l 27-hydroxylase gene transcription, and that this effect is mediated through a direct action of the hormone on the hepatocyte. These result s may provide an explanation for the increased bile acid pool and excr etion as found in humans with untreated diabetes mellitus and in exper imental animals with insulin deficiency.